This invention relates to a method and an apparatus for testing a sample for adenosine phosphates by causing the sample to react with a luminescent reagent containing an ATP regenerating enzyme.
It is known that stains produced by bacteria or food can be detected by detecting adenosine triphosphate (ATP) by a luminescent reaction using an appropriate reagent with luciferase. It, however, produces only a small output of light and it has hitherto been usual to use a photomultiplier for a photodetector to obtain a measurable output. The photomultiplier, however, requires a transformer and safety measures, since it needs a high voltage. Therefore, the apparatus as a whole is undesirably large and expensive.
There is also known a photodetector employing an avalanche photodiode (WO90/04775). The apparatus, however, requires a temperature stabilizing system for the avalanche photodiode which necessitates a working temperature of 0 to 5 deg. C. The system has a heat pump based on the Peltier effect and installed adjacent to the photodiode and is supplied with an electric current from a temperature control circuit to cool the photodiode. Therefore, the apparatus is undesirably complicated and expensive.
Therefore, we, the inventors of this invention, have paid attention to silicon photodiodes which do not require any high voltage or current source, and are not affected by temperature. The silicon photodiodes are, however, relatively low in sensitivity and do not always detect ordinary bioluminescence easily.
Some of us have proposed an invention entitled xe2x80x9cA bioluminescent reagent and a method of assaying for adenosine phosphates by using the reagent and a method of assaying for substances concerning an ATP transformation system by using the reagentxe2x80x9d as disclosed in Japanese Patent Laid-Open Publication No. HEI-9-234099. This invention is a method for detecting adenosinephosphates by a luminescent reaction of high sensitivity employing as a bioluminescent reagent at least a reagent containing an ATP regenerating enzyme, such as pyruvate orthophosphate dikinase, EC 2. 7. 9. 1. (hereinafter referred to as PPDK).
After further research work, we have discovered that adenosine phosphates, or stains can be detected by a simple apparatus if they are detected by measuring the quantity of light produced by the phosphates as a result of their reaction with a luminescent reagent containing an ATP regenerating enzyme, such as PPDK, and if the quantity of light is measured by a silicon photodiode.
According to a first aspect of this invention, there is provided a method for testing a sample by luminescence, which method comprises the steps of reacting the sample with a luminescent reagent containing an ATP regenerating enzyme to cause adenosine phosphates to produce light, and measuring the quantity of such light by a silicon photodiode.
According to a second aspect of this invention, there is provided an apparatus for testing a sample by luminescence, which apparatus comprises an inspecting device housed in a housing chamber, a silicon photodiode for receiving light from the inspecting device, and an operating system for processing the output signal of the photodiode to express the quantity of such light numerically.
More particularly, there is provided an apparatus for detecting stains, which comprises a chamber for housing an inspecting device for taking a sample of stains and causing them to produce light, a silicon photodiode for receiving such light, an operating system for determining the quantity of such light, a control panel and a display panel.
A silicon photodiode is a semiconductor device which responds to even a low level of light and outputs a measurable electrical signal, though it may be somewhat less sensitive to light than a photomultiplier. It does not require any high voltage or current source for its circuit, but is operable with a battery. It does not require any temperature stabilizing device, either, since it is less likely to be affected by any temperature variation than any other diode, such as an avalanche photodiode. Moreover, it is stronger than a photomultiplier, and is not adversely affected by exposure to intense light. Therefore, the apparatus of this invention for testing a sample for cleanness is by far smaller in size, lighter in weight and less expensive than any known apparatus.
The luminescent reagent used for the purpose of this invention contains an ATP regenerating enzyme, such as PPDK, and may be used with luciferin, or luciferase. It emits light and maintains a high level of stable luminescence by reacting not only with ATP, but also with adenosine diphosphate (ADP), adenosine monophosphate (AMP) or ribonucleic acid (RNA). Thus, it makes up for the light sensitivity of the silicon photodiode which is somewhat lower than that of the photomultiplier.
The stains in the context of this invention include adenosine phosphates, such as ATP, ADP, AMP and RNA or the like, and preferably refer to stains detected by a cleanness test.
The ATP regenerating enzyme in the context of this invention may be any enzyme catalyzing the reaction for forming ATP from AMP. Examples are phosphoenolpyruvate synthetase, EC 2.7.9.2., and a combination of adenylate kinase, EC 2.7.4.3. and pyruvate kinase, EC 2.7.1.40. PPDK is, however, preferred.
The PPDK used for the purpose of this invention is an enzyme catalyzing the reaction for forming ATP, pyruvic acid and phosphoric acid by acting upon AMP, phosphoenolpyruvic acid and pyrophosphoric acid, respectively, in the presence of a magnesium metal ion. It is easily available, as its physical and chemical properties and processes for its manufacture are already known (see Japanese Patent Laid-Open Publication No. HEI-9-234099).
The luminescent reagent containing an ATP regenerating enzyme may be prepared if, for example, PPDK is added to a luminescent reagent containing luciferin, luciferase and a metal salt, and a still more effective reagent may further contain phosphoenolpyruvic and pyrophosphoric acids. It detects even a small amount of stains by responding not only to ATP as an indicator of stains, but also to AMP. A reagent still further containing an enzyme catalyzing the reaction for forming ATP from ADP and/or an enzyme decomposing RNA detects a still smaller amount of stains by responding not only to ATP and AMP, but also to ADP and RNA.
This invention may be carried out by applying a swab to wipe stains off the surface to be tested, and dipping it in an extraction reagent containing a surface active agent for extracting adenosine phosphates, such as ATP, ADP, AMP and RNA, from stains, such as bacteria, whereby a sample solution is obtained. It is mixed with a luminescent reagent containing an ATP regenerating enzyme, such as PPDK or the like, and the quantity of light emitted by their mixture is measured by a stain testing apparatus, whereby the stains on the surface to be tested are detected. It is preferable to use a cleanness or hygiene monitoring device including the swab and the extraction and luminescent reagents as a unitary set to make the inspection still easier.
A hygiene monitoring or wipe inspecting device including a luminescent reagent containing an ATP regenerating enzyme, such as PPDK, makes a small, lightweight and economical apparatus which ensures a very accurate and reliable test for cleanness. More specifically, the surface to be tested is wiped by a swab in the hygiene monitoring device, the swab is dipped in the extraction reagent in the device, the resulting sample solution is reacted with the luminescent reagent held in the device and containing an ATP regenerating enzyme, such as PPDK, the hygiene monitoring device as a whole is set in the cleanness testing apparatus, and the quantity of light thereby emitted is measured to determine the amount of stains.
According to this invention, the use of a luminescent reagent containing an ATP regenerating enzyme, such as PPDK, ensures a stable emission of intense light detectable even by a silicon photodiode, and thereby makes it possible to realize a small and lightweight testing apparatus.